Project Summary/Abstract Multiple Myeloma (MM) is the second most common form of blood cancer and remains an incurable and deadly disease. Proteasome inhibitor (PI) therapy is a cornerstone in the treatment of MM, but resistance to this class of agent is an emerging challenge in the clinic. New therapeutic approaches that specifically target resistance are needed to maximize responses and ultimately produce cures. We originally identified hit-stage compound, E61, that selectively killed MM cells over normal cells, restored the activity of PIs in resistant MM cells, and showed early signs of anti-MM activity in a mouse model without any signs of toxicity. In the Phase 1 component of the project we engaged in a medicinal chemistry program that delivered a significantly more potent lead molecule, E64FC26, with improved drug-like properties, robust in vivo efficacy, and a strong patent position. Furthermore, Phase 1 identified protein disulfide isomerase (PDI) as the molecular target of this new class of small molecule. PDI regulates protein folding and is therefore a rational drug target for MM, which is a cancer that produce mass amounts of protein and is highly sensitive to disruption in protein homeostasis. Despite this rationale, no PDI inhibitors have advanced to clinical trials in humans, primarily due to limitations in the in vitro PDI assays that are used in drug discovery. The specific goals of the Phase 2 project are (1) to complete the chemical optimization of E64FC26 to further improve its pharmaceutical properties and enhance oral bioavailability, (2) to demonstrate the anti-MM efficacy of optimized E64FC26 in multiple mouse models of MM, and (3) to conduct dose range finding/maximum tolerated dose determining studies along with toxicokinetics in rats. These study aims are based on strong preliminary data and a large-scale medicinal chemistry program that identified key structure activity relationships (SAR) and a potent PDI inhibitor. This information has enabled the strategic design of a short-list of derivatives that we will test in the proposed study. Industry standard ADME assays and pharmacokinetics will be used to evaluate new E64FC26 derivative, and established mouse models of MM that recapitulate the human pathology and predict clinical efficacy will be used. Industry standard dose range finding studies will facilitate formal GLP-compliant toxicology that will enable an IND application to the FDA. Through the use of these tools, and predictions based on a breadth of preliminary data, it is our expectation that this work will deliver a promising new drug candidate for the treatment of refractory MM.